Routine TEM Staining Protocol for Tissues

 

Fixation:

 

1.       Fix 1 mm tissue blocks in 4% formaldehyde and 1% glutaraldehyde in 0.1 M PB (pH 7.4) for at least 2 hours to overnight.

2.       Immerse in 8% (0.2M) sucrose in 0.1 M PB 3x15min or overnight.

3.       Post-fix in 1% osmium tetroxide in 0.1 M PB 1 hour.

 

Dehydration:

 

4.       50% ethanol  15min

5.       70% Ethanol  15min

6.       95% Ethanol  15min

7.       100% Ethanol  2x15min

8.       100% Propylene oxide  2x15min

9.       1:1 Epon and Propylene Oxide for1-2 hour.

10.    2:1 Epon:Propylene Oxide overnight in dessicator with top off.

 

Embedding:

 

11.    Embed in beam capsules.

12.    Bake in 60 C oven for 48 hours.

 

Sectioning:

 

13.    Semithin section and Toluidine blue stain for 2-5 min

14.    Observe sections under microscope for precise location to cut for ultrathin sections

15.    Ultrathin sections at 60-90 nm thick (silver-yellow color) and collect sections onto grids. Dry sections overnight before staining.

 

Staining:

 

16.    Stain grids with uranyl and lead citrate.

17.    Observe under electron microscope.

 

Solutions and Reagents:

 

0.2M PB, pH 7.4:

Na₂HPO4 ------------------------ 21.8 g

NaH₂PO4 ------------------------ 6.4 g

Distilled water ------------------ 1000 ml

 

4F1G Fixative (4% Formaldehyde & 1% Glutaraldehyde in 0.1 M PB, pH 7.4):

0.1M PB, pH 7.4 --------------- 88 ml

37-40% Formaldehyde ----- 10 ml

50% Glutaraldehyde --------- 2 ml

 

8% (0.2M) Sucrose in 0.1M PB:

Sucrose ------------------------- 8 g

0.1 M PB ------------------------ 100 ml

 

1% Osmium in 0.1M PB:

2% Osmium -------------------- 5 ml

0.2M PB, pH7.4 ---------------- 5 ml

 

5% Uranyl Acetate Solution:

To prepare 50 ml, add 2.5 g of uranyl acetate to 50 ml of distilled water. Cover with foil and stir overnight. Add 10 drops of glacial acetic acid. Store solution at 4 C. This solution is stable for at least 6 months at 4 C.

 

Reynold’s Lead Citrate Solution:

To prepare 50 ml, add chemicals in distilled water in following order

Lead nitrate ------------------------------- 1.33 g

Sodium citrate, dihydrate -------------- 1.76 g (solution becomes cloudy when sodium citrate is added)

1N NaOH ----------------------------------- 5 ml  (solution becomes clear when NaOH is added)

Distilled water ----------------------------- 30 ml

Stir for 10 minutes to dissolve and add additional 15 ml of distilled water. Store solution for 3-6 months at 4 C. Note: the mount of NaOH is very important. Too much will make solution cloudy.

 

Epon 812:

 

Final Volume	EM bed-812	DDSA	NMA	DMP-30 (2%)
20.91 ml	10 ml	4.5 ml	6 ml	0.41 ml
31.37 ml	15 ml	6.75 ml	9 ml	0.62 ml
41.82 ml	20 ml	9 ml	12 ml	0.82 ml
52.28 ml	25 ml	11.25 ml	15 ml	1.03 ml

       Mix all four components in plastic beaker and stir with wood stick.